1. Field of the Invention
The present invention relates to methods and systems for detecting a member of a binding pair with surface plasmon resonance.
2. Background
When two media of different refractive index are joined at an interface, light from the medium having a higher refractive index can be partly reflected and partly refracted at the interface. If the angle of incidence is above a certain critical angle, the light is entirely internally reflected and no light is refracted across the interface. At angles above the critical angle for which total reflectance is observed, an electromagnetic field component of the light penetrates a distance on the order of tens of nanometers into the medium of lower refractive index. The electromagnetic field creates an exponentially detenuating evanescent wave.
If the interface between the two media is coated with a thin layer of metal, the intensity of the reflected light is reduced at a specific incident angle due to resonance energy transfer between the electromagnetic field and surface plasmons of the metal. The resonance energy transfer is referred to as surface plasmon resonance. Modifications to the surface of the thin metal film can influence the resonance conditions of the metal.
Surface plasmon resonance can be used as a technique used for the detection of binding to surface layers and can provide information characterizing the properties of a thin film. One example of surface plasmon resonance involves attaching a receptor to a surface of a sensor device. The sensor device includes a thin metal layer to which the receptor is attached. Gold is typically used as the metal layer. One side of the metal layer is contacted with a solution sample containing an analyte of interest. The binding of the analyte with the receptor causes changes in the optical characteristics of the opposite surface of the metal layer. Measuring the changes in optical characteristics can indicate whether binding of the analyte with the receptor occurred.
The absorptive transition that arises at the resonance condition in a metal is commonly referred to as a plasmon resonance band. A metal behaves as a conductor below the plasmon resonance frequency and as an insulator above the plasmon resonance frequency. At the resonance frequency, the metal absorbs electromagnetic radiation.
One theoretical model for describing the intense absorption observed in plasmon resonance is the Drude model. The Drude model is based on an assumption that a metal is a regular three-dimensional array of atoms or ions with a large number of electrons free to move about the whole metal. The “sea of electrons” can be treated as a mass, and the electrostatic attraction of the metal lattice as a spring. The electromagnetic radiation that impinges on the metal surface is a forcing term. The plasmon resonance condition is analogous to the resonance condition that arises for any forced harmonic oscillator. The plasmon resonance frequency is given by Equation 1.                               ω          p          2                =                              n            ⁢                                                  ⁢                          e              2                                            m            ⁢                                                  ⁢                          ɛ              0                                                          (        1        )            where n is the free charge carrier concentration, m is the free electron mass, e is the elementary charge of an electron, and ∈0 is the permittivity of vacuum. Wooten, F. Optical Properties of Solids; Academic Press, Inc.: Sandiego, 1972.
A metal typically has a relatively high charge carrier concentration that is not altered by the application of light and/or an electric field. At the plasmon resonance frequency, free electrons of a metal will oscillate and absorb energy at a certain angle of incident light. The angle of incident light when surface plasmon resonance occurs is typically referred to as the surface plasmon resonance angle. The surface plasmon resonance of noble metals such as gold and silver are much lower than those of other metals and are accessible by visible and UV spectroscopic approaches. Gold and silver exhibit sensitivity of the surface plasmon frequency and the wave vector. The angular condition for coupling the electromagnetic radiation and surface plasmon as observed in the optical absorption of the plasmon resonance band has been used to detect the binding of surface layers for p-polarized radiation. The change in index of refraction at the surface of the metal changes the angle for maximum plasmon absorption. However, the changes in angle are typically on the order of millidegrees and detection can be difficult. Changes in wavelength observed in gold and silver are typically even more difficult to detect.
Current methods for detecting analytes on surface layers measure the change in index of refraction on a metal surface by measuring the change in the angle of maximum absorption (minimum reflectance). One common implementation of the detection method is the use of a HeNe laser at 632.8 nm in a fixed wavelength measurement. The change in HeNe intensity is proportional to the change in index of refraction of the gold. Current methods detect a change in the surface plasmon resonance angle. For example, U.S. Pat. No. 6,127,183 proposes the detection of changes in the minimum angle of plasmon resonance on gold. U.S. Pat. No. 5,641,640 proposes the detection of changes in the surface plasmon resonance angle to detect analytes bound to the surface of a metal.
Such surface plasmon resonance detection systems may have the sensitivity to detect the binding of an analyte to a submonolayer of surface receptors or binding sites. Hydrogels such as carboxymethylcellulose have been used as an intermediary between the receptors or binding sites to increase the sensitivity of detection. However, the additional material on the surface may present several problems. The direct calibration of the signal with binding events may become more difficult because any effect that swells the hydrogel may give rise to a false signal change. This has led to the use of kinetic assays such as those proposed in U.S. Pat. No. 6,143,574. However, kinetic assays may require that all of the binding sites in the hydrogel be uniformly accessible.
Typical spectra for gold are shown in FIGS. 1A and 1B. FIG. 1A shows the spectrum of reflectivity or light intensity (I) as a function of angle (θ) detected prior to binding on a gold optical layer. There is a sharp decline in intensity at the plasmon resonance angle θ1. FIG. 1A shows the spectrum of light intensity (I) as a function of angle (θ) after binding occurs between the binding pair on a gold optical layer. The sharp decline in intensity at the plasmon resonance angle θ2 is observed. The change in angle between θ1 and θ2 indicates a change in the optical layer due to binding events.